Capacitation is a biochemical process that mature spermatozoa undergo in the upper female reproductive tract, within the oviduct and uterus, that allows for the beginning of sperm activation. It is also known as cellular-implantation or just implantation. Capacitation is the penultimate step of spermatozoa activation, with the acrosome reaction following it. Sperm must undergo capacitation and the acrosome reaction in order to penetrate through the cumulus ooporus and the zona pellucida of an oocyte. Capacitation specifically allows for induction of hyperactivation and hypermotility, processes that set up for oocyte penetration. Capacitation is also necessary for chemotactic swimming through the female reproductive tract, where progesterone gradients guide the sperm to the egg [1] . Compounds such as heparin and progesterone can be used to induce capacitation.
As a result of the sperm entering the upper female reproductive tract, the sperm are introduced to an extracellular environment that contains a cholesterol acceptor (usually serum albumin), electrolytes such as sodium (Na+), potassium (K+), chloride (Cl-), bicarbonate (HCO3-), magnesium (Mg2+), calcium (Ca2+), and phosphate (PO43-), and energy substrates such as glucose, pyruvate, and lactate [2] . For purposes of in vitro fertilization, capacitation occurs by incubating spermatozoa that have been retrieved via ejaculation or extracted from the epididymis and incubated in a defined medium for several hours. There are different techniques to perform the capacitation step: simple washing, migration (swim-up), density gradients, and filter. The objective is to isolate as many motile spermatozoa as possible and to eliminate non-motile or dead spermatozoa.
Mammalian sperm membranes have an approximate lipid composition of 70% phospholipids, 25% cholesterol, and 5% glycoproteins, though the exact percentages are species dependent. A notable distinguishing characteristic between mammalian species is the variable ratio of cholesterol to phospholipid (C/PL) in the plasma cell membrane. The duration of capacitation has been shown to be related to the C/PL ratio of sperm membranes, contributing to the difference in capacitation duration between species [3] . Studies have reported that sperm capacitation in humans takes 3-10 hours, on average.
The majority of capacitation studies have been conducted on mice as they are a sufficient de facto surrogate model, but limitations to these models do exist [4] . For instance, in mice, ejaculate is directly deposited into the uterus while, for humans, it is in the vaginal canal [3] . Additional anatomical and physiological differences between species also contributes to the potential inaccuracies of using mice for studying capacitation. Despite this, the use of animal models has contributed a significant amount to our understanding of capacitation.
Non-mammalian spermatozoa do not require activation via capacitation and the acrosome as they are ready to fertilize an oocyte immediately after release from the male.
Sperm capacitation consists of five main steps: changes to the sperm plasma membrane that cause increased membrane fluidity, pH changes, ion flux, activation of the cAMP-PKA pathway and downstream phosphorylation, and changes to membrane potential (hyperpolarization) which result in hyperactivation and consequent enhanced motility of sperm [5] .
Capacitation has two effects: destabilisation of the acrosomal sperm head membrane which allows it to penetrate the outer layer of the egg, and chemical changes in the tail that allow a greater mobility in the sperm. [6] The changes are facilitated by the removal of sterols (e.g. cholesterol) and non-covalently bound epididymal/seminal glycoproteins. The result is a more fluid membrane with an increased permeability to Ca2+ ion.
An influx of Ca2+ produces increased intracellular cAMP levels and thus, an increase in motility. Hyperactivation coincides with the onset of capacitation and is the result of the increased Ca2+ levels. It has a synergistic stimulatory effect with adenosine that increases adenylyl cyclase activity in the sperm.[ citation needed ]
The tripeptide fertilization promoting peptide (FPP) is essential for controlling capacitation. FPP is produced in the prostate gland as a component of the seminal fluid. FPP comes into contact with the spermatozoa during ejaculation, as the sperm and seminal fluid mix. High levels of active FPP prevent capacitation. After ejaculation, the concentration of FPP drops in the female reproductive tract. [ citation needed ]
Because assisted reproductive technologies, or ARTs, such as in vitro fertilization (IVF) or intrauterine insemination (IUI) require the induction of sperm cell capacitation outside of normal biological parameters, numerous methods have been developed to induce this process in mammalian sperm cells. Sperm cells are harvested through ejaculation or harvested from the caudal epididymis and allowed to liquefy at room temperature. Capacitation can then be induced by adding media designed to mimic the electrolytic composition of the fallopian tubes, where fertilization occurs. These media vary between species, but are saline-based and contain energy substrates such as lactate, pyruvate, and possibly glucose. A cholesterol acceptor is required to facilitate the removal of cholesterol from the sperm cell membrane, which is often albumin. Bovine serum albumin is typically used for in vitro animal studies, and human serum albumin (HSA) is used in human sperm capacitation induction.
Bicarbonate is a vital component of capacitation-inducing media, as it is co-transported into the cytosol where it activates soluble adenylyl cyclase (sAC) as well as acts as a pH buffer necessary to prevent decreasing the pH in the culture, a necessary addition when incubating cells at 5% CO2 as is generally used although not required. Calcium chloride is added to facilitated the influx via of calcium cations. [7] [8] In animal models, Tyrode's albumin lactate pyruvate (TALP) medium is typically used as a base, which contains each of these components. In humans, human tubal fluid (HTF) is used.
These media can be supplemented with other chemicals to induce hyperactivated sperm motility and/or the acrosome reaction. For animal in vitro fertilization, caffeine at 5 mM concentration is a strong inducer of sperm capacitation in vitro. [9] [10] Calcium ionophores are also ideal to induce capacitation. [10] Adding heparin to capacitation inducing medium mimics the secretion of heparin-like gycosaminoglycans (GAGs) near the oocyte and initiates the acrosome reaction. This effect is magnified when adding lysophosphatidylcholine (LC) in conjunction with heparin. [11] Catecholamines such as norepinephrine at low concentrations have been shown to assist in acrosome reaction induction. [12]
The traditional methods to perform in vitro capacitation are:
PICSI, MACS or microfluidic chips are more recent methods that can be used to induce capacitation in vitro.
Numerous methods have been developed to assess the degree to which sperm cells are undergoing capacitation in vitro. Computer-aided sperm analysis (CASA) was developed in the 1980s for measuring sperm kinematics. [14] CASA uses phase-contrast microscopy combined with sperm tracking software to analyze sperm motility parameters. [14] Certain parameters such as curvilinear velocity (VCL), straightline velocity (VSL), average path velocity (VAP), and the amplitude of lateral head displacement (ALH) have been shown to be positively correlated with the acquisition of fertilization competency and are thus used to identify hyperactive sperm cell motility. [15]
While motility measurements are critical for identifying the presence of hyperactive motility, additional methods have been developed to identify the occurrence of the acrosome reaction. A simple method uses Coomassie brilliant blue G250 to stain cells, providing visual evidence of intact or reacted acrosomes. [16] More advanced techniques employ fluorescent or electron microscopy methods. Fluorescein-conjugated Peanut agglutinin (FITC-PNA) or Pisum sativum agglutinin (FITC-PSA) can be used to fluorescently tag the acrosome of sperm cells, which can be then used to assess the status of the acrosome using a fluorescent microscope. [17] [18] [19]
The discovery of this process was independently reported in 1951 by both Min Chueh Chang [20] and Colin Russell Austin. [21] [22]